穿梭质粒pCEPmB_7的构建及其对小鼠宫颈癌U_(14)细胞体内生长的影

Construction of Shuttle Vector pCEPmB 7 and Its Effect on Growth of Mouse Cervical Cancer Cell U 14 in Vivo

目的：构建可用于肿瘤基因治疗研究的基因表达载体ｐＣＥＰｍＢ７，并研究该质粒转染小鼠宫颈癌Ｕ１４细胞后的体内致瘤性变化。方法：（１）用限制性核酸内切酶ＨｉｎｄⅢ和ＸｈｏⅠ分别对质粒ｐＣＥＰ４和ｐＬＸＳＮｍＢ７进行双酶切，分别回收１０．４ｋｂ的线性ｐＣＥＰ４ＤＮＡ片断和０．９ｋｂ的ｍＢ７ｃＤＮＡ片断，将上述两片断混合，在Ｔ４－ＤＮＡ连接酶的作用下连接，连接液转化受体菌ＴＧ－１，琼脂糖凝胶电泳鉴定连接是否正确；（２）用电穿孔法将ｐＣＥＰｍＢ７转染Ｕ１４细胞，转染后的细胞经ＨｙｇｒｏｍｙｃｉｎＢ（２００μｇ／ｍｌ）选择培养２周后得到选择阳性混合克隆细胞Ｕ１４／ｐＣＥＰｍＢ７，用流式细胞仪检测Ｕ１４／ｐＣＥＰｍＢ７细胞中Ｂ７表达阳性细胞的百分率；（３）将Ｕ１４和Ｕ１４／ｐＣＥＰｍＢ７细胞分别以不同的剂量皮下接种昆明鼠，找出两种细胞的最小成瘤剂量（ｍｉｎｉｍａｌｔｕｍｏｒｉｇｅｎｉｃｄｏｓｅｓ，ＭｉＴＤ）。结果：（１）连接后的新质粒经双酶切和单酶切后，琼脂糖凝胶电泳鉴定连接正确，该质粒即为ｐＣＥＰｍＢ７；（２）流式细胞仪检测结果显示，Ｕ１４细胞的Ｂ７表达阳性率为５．２４％，Ｕ１４／ｐＣＥＰｍＢ７细胞的Ｂ７表达阳性率为４?

Objective:To construct the gene expression vector pCEPmB 7 used for tumor gene therapy,and to study the changes in tumorigenicity of mouse cervical cancer cell U 14 transfected by this vector in vivo.Methods:(1)The plasmids pCEP 4 and PLXSNmB 7 were digested respectively by restricted endonuclease Hind Ⅲ and XhoⅠ,the DNA fragments of 10.4 kb linear pCEP 4 and those of 0.9 kb mB 7 were retrieved,mixed and linked under T 4 DNA liqase reaction.The ligation liguid transformed receptor bacterium TG 1 and ligation was appraised by agarose gel electrophoresis.(2)U 14 cells were transfected by pCEPmB 7 through electroporation.Transfected cells were selectively cultured for two weeks with hygromycin B at 200 ug/ml concentration,the selected positive mixed clone cells U 14 /pCEPmB 7 were obtained.The percentage of B 7 positive expression cells in U 14 /pCEPmB 7 was determined by flow cytometry.(3)To inoculate Kunmin mice hypodermically with U 14 and U 14 /pCEPmB 7 at different dosages separately to find out the minimal tumorigenic dose (MiTD) of the two cells.Results:(1)The new plasmid after ligation was digested with two enzymes and one enzyme,and confirmed by agarose gel electrophoresis to be linked correctly was pCEPmB 7.(2)The flow cytometry test showed the percentage of B 7 positive expression was 5.24% in U 14 48.52% in U 14 /pCEPmB 7.(3)The MiTD of U 14 was 4×10 4 cells and that of U 14 /pCEPmB 7 was 4×10 5 cells.Conclusion:When U 14 was transfected by pCEPmB 7,the percentage of positive expression of costimulatory molecule B 7 and MITD of mice in vivo increased distinctly,indicating the tumorigenicty of U 14 /pCEPmB 7 decreased and its immunogenicity enhanced.It is suggested that the shuttle vector pCEPmB 7 may be used for preparing tumor gene vaccine.